We will investigate further the mechanism by which freezing injures living cells, concentrating primarily on the effects of osmotic volume reduction and associated membrane stresses which we have proposed lead to membrane injury. Both biochemical and electron microscopic studies of the membranes of cells subject to osmotic volume reduction will be undertaken to determine whether alterations in structure of lipid content result. Past studies of freezing have emphasized the role of colligative cryoprotectants and, especially, the obstacles associated with the introduction and removal of penetrating agents. These studies have led to new insights into these procedures and have enabled the design of protocols for cryoprotection that do not in themselves threaten cell integrity. These protocols will be applied to the cryoprotection of rabbit and dog kidneys and the relationship between cryoprotectant concentration, freezing temperature, and duration of storage will be evaluated.